DESCRIPTION: Innate cellular antiviral defenses are likely to influence the outcome of infections by many human viruses, including hepatitis C virus (HCV), a positive strand RNA virus that frequently establishes persistent infections leading to chronic hepatitis, cirrhosis and liver cancer. However, little is known about how hepatocytes sense HCV infection and initiate protective responses. We have recently shown that non-neoplastic PH5CH8 hepatocytes contain two distinct antiviral signaling pathways, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I), to recognize viral double-stranded (ds) RNA and lead to subsequent interferon antiviral response. Our long-term goal is to elucidate the role of these signaling pathways in hepatocellular control of HCV infection. While RIG-I signaling was recently shown to contribute to sense intracellular HCV RNA replication, the role of TLR3 signaling in recognition and control of HCV infection remains to be determined, as previous investigations were all conducted in hepatoma Huh7 cells which lack a functional TLR3 pathway. We hypothesize that TLR3 signaling is an important antiviral mechanism of hepatocytes, and contributes to host defenses against HCV. We propose three aims for this application: (1) Determine the subcellular localization of TLR3 and the mechanism of TLR3 signaling in hepatocytes where it is initiated and to what extent it cross-talks with the RIG-I pathway. (2) Determine whether structured HCV RNA and HCV infection activate TLR3 signaling pathway in TLR3 reconstituted Huh cells and whether TLR3 is associated with HCV replication complex in cells supporting HCV replication. (3) Determine whether TLR3 signaling contributes to hepatocellular control of HCV replication. This proposed research shall advance our knowledge regarding virus-host interactions in hepatitis C pathogenesis and the role of innate immunity in controlling HCV infection, which would potentially benefit the design of new therapeutic interventions for HCV infection.